Vaccines are widely used for the prevention and/or therapy of many different diseases. Vaccine utilizing polysaccharide (PS) alone has relatively low immunogenicity. To overcome the relatively low immunogenicity of polysaccharide, PS vaccines are conjugated to protein carriers to increase immunogenicity and provide long-term protection in young children. Many conjugate vaccines are already approved and marketed throughout the world. Examples of such vaccines, known as “conjugates” are available for Haemophilus influenzae type b (e.g., ACTHIB® [Haemephilus b conjugate vaccine, Tetanus toxoid conjugate], HIBERIX [Hamemophilus b conjugate vaccine, Tetanus toxoid conjugate]), Neisseria meningitides types A, C, W and Y (e.g., MENACTRA® [Meningococcal (Groups A, C, y and W135) Polysaccharide Diphtheria Toxoid Tetanus conjugate vaccine]) and S. pneumoniae (e.g., PREVNAR® [Pneumococcal 7-valent conjugate vaccine (Diphtheria CRM197 Protein], SYNFLORIX® [Pneumococcal conjugate vaccine (Non-Typeable Haemophilus influenza (NTHi) protein D, Diptheria or tetanus toxoid conjugates]).
To extend the shelf-life of conjugate vaccines, formulations are often lyophilized. Lyophilization of conjugate, however, can lead to protein aggregation during freezing and storage. As aggregation increases, the effective concentration of available immunogen decreases this may result loss of immunogenicity. Also the lyophilization processes involving freezing and sublimation have stresses which result into chemical degradation which subsequently releases free polysaccharide.
Disaccharides are used as stabilizers during the process of lyophilization. The role of a disaccharide is to provide water replacement and subsequent hydrogen bond formation with polysaccharide protein conjugate. Also disaccharides form sugar glasses of extremely high viscosity. The conjugate and water molecules are immobilized in the viscous glass, leading to extremely high activation energies required for any reactions to occur. The role of crystalline glass former with sugar is to form glassy matrix with the conjugate or mixture of conjugates arresting any degradation.
The polysaccharide component of conjugate vaccines undergoes gradual depolymerization at a rate that depends on the type of conjugate, formulation components and storage conditions. This causes reduction in molecular size of the polysaccharide component, i.e. an increase in free polysaccharide. Hence tests should be conducted to ensure stability of product.
The immunogenicity of a Polysaccharide-protein conjugate vaccine is decreased by the presence of uncoupled Polysaccharide, wherein the magnitude of this effect seems to be Polysaccharide type dependent. Refer Rodriguez et al (1998), “Immunogenicity of Streptococcus pneumoniae type 6B and 14 polysaccharide-tetanus toxoid conjugates and the effect of uncoupled polysaccharide on the antigen-specific immune response,” Vaccine, 16(20), 1941-1949.
Polysaccharide-carrier protein conjugates are known to release free polysaccharide after conjugation while further processing, lyophilization or storage in liquid as well as solid formulations. Clinical protection is conferred only by polysaccharide that is covalently bound to the carrier protein. Accordingly vaccines demonstrating adequate immunogenicity and thermostability contain amounts of unbound polysaccharide ranging from less than 10% of the total polysaccharide, refer WHO TRS, pg 47-48, No. 897, 2000.
Also the average residual moisture content of less than 2.5% is desired, refer WHO TRS, pg 45, No. 897, 2000. Further accelerated and real-time studies are additional parameters that provide supporting evidence for stability.
U.S. Pat. No. 5,618,539 describes stabilized viral vaccines, particularly against polio, comprising an aqueous solution of live virus and a stabilizing compound, which has at least two amino or imino groups, such as basic amino acids (e.g. lysine, arginine etc.). This patent mentions also that the stabilizing compound allows a thermal stability increase of the virus in relation to stabilized viruses by magnesium chloride (MgCl2) (another stabilizing agent). But even with the use of effective stabilizers, as magnesium chloride or amino acids, nothing stops the vaccine potency loss, if it is thawed during the transport or storage. Rombaut et al. (B. Rombaut, B. Verheyden, K. Andries and A. Boeye).
WO 2009/111849 A1 discloses a method for producing a stabilized polio virus vaccine composition in liquid form by applying high hydrostatic pressure.
U.S. Pat. No. 5,728,386 discloses a process for making thermostable varicella zoster virus vaccine, wherein the lyophilized or liquid preparation of live attenuated VZV was heated under highly inactivating conditions.
WO/1998/028000 discloses thermostable vaccine formulations comprising an increased amount of a 6-carbon polyhydric alcohol (such as sorbitol), an increased amount of a disaccharide (such as sucrose) and an amount of a physiologically active buffer to adjust the pH from about 6.0 to about 7.0.
EP1123710 discloses a thermostable Hepatitis A vaccine formulation which can be stored at ambient temperature for a longer time.
WO/2001/041800 discloses a method for stabilizing a MenC vaccine upon lyophilization comprising: (a) dissolving the MenC vaccine in a dissolution buffer comprising at least one amorphous excipient (sucrose) and an amorphous buffer (histidine) to form a mixture, and (b) lyophilizing the mixture.
Despite these advances in the area of vaccine formulations, there remains a distinct need for conjugate vaccine formulations with improved thermostability, shelf-life and immunogenicity especially meningococcal and pneumococcal conjugate vaccines. None of the prior art stabilizers impart the desired enhanced sustained level of stability. The present invention addresses and meets the long felt need for a stabilizer and conjugate vaccine formulation with increased thermostability subsequent to lyophilization.
The present invention arises from the surprising discovery that it is possible to prepare lyophilized polysaccharide protein conjugate vaccines by utilizing a specific stabilizer combination of amorphous excipients and buffer resulting in improved thermostability and immunogenicity.